Abstract:Abstract:Actinomycetes can produce numerous secondary metabolites with novel structures and unique bioactivities，which are significant for pharmaceutical industry，agriculture and environmental protection． Whole-genome sequencing data demonstrate that actinomycetes contain plenty genes coding for transporters with ATP-binding cassette (ABC)，which play important roles in nutrient uptake，secondary metabolite export，xenogenous toxin detoxification，and so on． In this review，the structures and mechanisms of the ABC transporters were described． We also comprehensively discussed research advances including ours on actinomycete ABC transporters，with emphasis on ABC exporters responsible for the secretion of secondary metabolites． Finally，research hotspots and application prospect of actinomycete ABC transporters were also addressed．

Abstract:Abstract:Anthrax，as a fulminating infectious disease，threatens human’s health seriously． Bacillus anthracis，the agent of anthrax，was classified into the second kinds of pathogenic microorganisms (one kind of the highly pathogenic microorganism) in the List of Human Pathogenic Microorganisms issued by the Chinese government． The spores formed by B． anthracis are potential material for biological warfare agent and biological terror． Therefore，it is very important and pressing to develop sensitive，efficient detection methods for the bacteria． For detection methods of B． anthracis，there are four types of targets:spores，vegetative cells，genes and anthrax toxin proteins． Among them，detection methods targeting spores and vegetative cells are developed． However，owing to disadvantages in specificity and clinical practicality，these methods are far from satisfaction． Detection methods targeting genes of B． anthracis are satisfactory in specificity and sensitivity，while it is short in clinical diagnosis． At the same time，the development of detection methods targeting anthrax toxin makes it possible to acquire information about main causative agent directly，which brings about great help in clinical diagnosis as well as epidemiology research． Herein，we summarized briefly detection methods of B．anthracis developed currently，investigated their application ranges and detection capacity，and discussed the development trend of related research，expecting favoring the profession developing detection methods of B．anthracis．

Abstract:Abstract:［Objective］We studied the isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris by using culture-dependent approaches． ［Methods］Fresh fecal samples of healthy Panthera tigris tigris were collected from Yunnan Safari Park． Pretreatment of the samples，isolation media and inhibitors were tested for actinobacteria isolation． 16S rRNA genes of actinobacteria were sequenced and subjected to phylogenetic analysis．［Results］The abundance of culturable actinobacteria was 1.10×108 cfu/g colony forming units (CFU) per gram of feces (wet weight)．We obtained 110 purified cultural actinobacterium strains．The analysis based on 16S rRNA gene sequences showed that these strains were distributed in 10 different families and 12 genera of actinobacteria at least，and most of them were non-filamentous，such as Arthrobacter，Dietzia，Kocuria，Corynebacterium and Microbacterium．Streptomyces was the mainly classical filamentous actinobacteria，and up to 64% of total．［Conclusion］ Drying and heating up the fecal samples can greatly increase the rate of the actinobacteria．Many kinds of inhibitors and chemical defined media are suitable for isolation of fecal actinobacteria． The culturable actinobacteria are abundant in Panthera tigris tigris feces.Our study found an effective method to isolate animals' fecal actinobacteria and it's useful for studying and exploiting animals' fecal actinobacteria．

Abstract:Abstract:［Objective］Large plasmid pCQ4 was detected in Streptomyces sp． W75 from Artemisia annua L． We clonined，sequenced，analyzed and characterized pCQ4． ［Methods］Southern hybridization was used to determine restriction map of pCQ4． To clone the full-length of pCQ4，conjugation and recombinational cloning in a BAC vector were used．［Results］ The complete nucleotide sequence of pCQ4 consisted of 84833-bp，encoding 129 ORFs which 40 ORFs resembled these of bacterial phages．W75 culture could infect W75 cured of pCQ4 and formed plaques on plate．Phage particle (ФCQ4) was observed by transmission electron microscopy． LinearФCQ4 DNA was detected on pulsed-field gel electrophoresis． Comparison to Streptomyces plasmid-phage pZL12，genes encoding major phage structural proteins resembled that of pCQ4．［Conclusion］Streptomyces plasmid pCQ4 could be transformed into lytic phageФCQ4，and the phage segment on pCQ4 might be a mobile unit．

Abstract:Abstract:［Objective］ Cotton leaf curl disease (CLCuD) is a major constraint to cotton production，causing great economic losses in Pakistan and India． In China，CLCuD has been discovered in the field of Nanning，GuangXi．To better understand this disease，we sequenced the virus-associated small DNA molecules．［Methods］ We purified total DNA from cotton and okra plants exhibiting leaf curl symptoms; PCR amplified and sequenced CLCuMV satellite DNA (DNAβ)-related small DNA molecules．［Results］ We identified 2 novel recombinant DNA molecules with 1384 nucleotides in cotton and 754 nucleotides in okra． The 1384 nt molecule contains partial DNA-A and DNAβ of CLCuMV GX1． It includes the intergenic region，adjacent AV2 and AC1 coding sequences，and reverse complementary AC3 of DNA-A and A-rich region of DNAβ．Common nucleotides were found around the junction points of DNA-A and DNAβ sequences，suggesting they were the sites of recombination．Comparison with previous reported CLCuMV recombinants produced in lab showed that the intergenic region of DNA-A and A-rich region of DNAβ were conserved on the recombination process． The 754 nt molecule was produced by deletion of CLCuMV DNAβ in the C1 open reading frame and A-rich region．［Conclusion］ We identified a novel recombinant molecule originated from CLCuMV DNA-A and DNAβ and a small defective molecule of DNAβ． This is the first report of sequence recombination and deletion of CLCuMV in China，which may be helpful to understand the CLCuMV evolution and host adaptation．

Abstract:Abstract:［Objective］To reveal the mechanism of AMPK signaling in the autophagy and apoptosis caused by troglitazone (TZ)．［Methods］To investigate the effect of TZ on alteration of autophagy and apoptosis in HeLa cells，fluorescence microscopy，electron microscopy，western-blotting，siRNA interference，flow cytometry and MTS assay were used．［Results］TZ attenuated AMP-activated protein kinase-α (AMPKα) phosphorylation，and stimulates autophagic process in HeLa cells．TZ induced the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II)，and degradation of sequestosome 1 (SQSTM1/p62)，two markers of autophagy，occurring prior to the caspase activation．Compound C，an AMPK inhibitor，increased basal and inhibits TZ-stimulated LC3-II content and TZ-depended PARP cleavage． Knockdown of the gene encoding autophagic proteins and AMPK conferred the cells resistance to apoptosis by TZ．［Conclusion］ Taken together，these data demonstrate that AMPK is involved in TZ promote autophagy，which precedes and contributes to caspase-dependent apoptosis．

Abstract:Abstract:［Objective］ Expression，purification and characterization of a laccase gene from Pleurotus ostreatus in Trichoderma reesei．[Methods] The strong promoter and terminator of cellobiohydrolase I (cbh1) gene from T． reesei were amplified by PCR and inserted into pBluescriptIISK(+) to form vector pSKCST． The laccase gene from Pleurotus ostreatus was de novo synthesized according to T．reesei condon bias and cloned into the vector pLacdt resulting in the expression vector pSKLDT．The linearized pSKLDT was introduced into T．reesei strain Tu6 by protoplast-mediated transformation． The screened laccase expression transformants were grown in shake flasks on minimal medium and the recombinant laccase was purified and characterized． ［Results］Transformants were isolated in selective screening medium plate and identified by PCR． The enzyme activity of laccase in transformant LC-7 was 237. 134 U /mL which was 28.6－fold higher than that in P． ostreatus． The specific activity of the purified enzyme was 9852 IU/mg． Enzymatic assay revealed that the optimum temperature for its activity was 50℃ and pH was 3. 0． The optimum substrate was ABTS and the Km and Vmax for ABTS were 7.58 ×10－2 mmol/L and 9.752×10－3 mmol/L/min． Metal ions like Cu2+，Zn2+，Fe3+，Mn2+，Ba2+，Mg2+and Fe2+had different inhibitory effect on purified laccase.［Conclusions］Under the regulation of cbh1 promoter and cbh1 signal peptide，heterologous laccase was successfully overexpressed in T． reesei．

Abstract:Abstract:［Objective］To construct a novel cell-surface display system of Thermomyces lanuginosus lipase (TLL) based on an efficient anchor protein Sed1p in Pichia pastoris，to screen recombinant strains with high enzyme activity and displaying rate，and further to characterize the enzyme． ［Methods］The lipase gene from T．lanuginosus was sub-cloned and fused with the anchor protein gene sed1 from Saccharomyces cerevisiae to construct a display vector pPICZαA- TLS．The vector pPICZαA-TLS was linearized by SacⅠand then transformed into P． pastoris GS115 by electroporation．After screening by tributyrin medium，a clone exhibiting the maximum lipase activity in shaking flask was chosen to treat with rabbit anti-FLAG-tag and R-PE-conjugated goat anti-rabbit IgG，and then its positive location on the cell wall was detected by fluorescence microscope and flow cytometer． The recombinant strain displaying TLL was further characterized as a whole-cell catalyst．［Results］A novel cell-surface display system of T． lanuginosus lipase was successfully established，and a clone with lipase activity of 257. 8U/g dry cells in shaking flask was obtained． The displayed TLL on the cell surface was confirmed by immunofluorescence，and the treated cells under the fluorescence microscope emitted brightly red fluorescence，and the displaying rate was 98. 36% detected by Flow Cytometer． The displayed TLL exhibited excellent thermostability and high tolerance to some organic solvents，and its maximal activity was observed at 30℃ and pH 8.0．The lipase activity was a little enhanced by K+，Ca2 + and Mg2 + and strongly inhibited by Cu2 + ，Mn2+ and Ni2+．However，ethylenediaminetetraacetic acid (EDTA)，Sodium lauryl sulfate (SDS) and Tween 20 showed little effect on the displayed TLL． ［Conclusion］The lipase TLL was successfully displayed on the cell surface of P． pastoris by the anchor protein Sed1p for the first time to obtain a whole-cell catalyst，which had high hydrolytic activity and excellent enzymatic characterization． Thus，we here established a solid foundation for industrial applications of the displayed lipase TLL．

Abstract:Abstract:Equol is the end metabolite of daidzein by certain intestinal bacteria，which has stronger bioactivity comparing with daidzein．［Objective］To evaluate the role of orally administrated equol-producing bacterium on increasing equol production and shifting intestinal microbiota of embracer animal．［Methods］In total 60 rats with or without ovariectomy at average bodyweight 211 ± 9g were divided into 5 groups． The differences on equol concentration and bacterial composition were compared in rats orally treated with distilled water，estradiol，daidzein，equol and daidzein + ZX7． ［Results］ The fecal equol concentration in daidzein groups was significantly higher than that in control and estradiol groups． The fecal equol concentration in daidzein + ZX7 groups was comparable to that in equol groups． Principal Components Analysis (PCA) of fecal DGGE profiles showed clear difference of fecal microbiota between ovariectomy done or undone rats． Fecal population of Bacteriodetes showed strong correlation with fecal equol concentration． ［Conclusion］Normal intestinal microbiota in rats might have the capacity to convert daidzein into equol． Dietary source of equolproducing bacterium ZX7 might have the possibility to increase the equol production of embracer animal． Endogenous estrogen concentration might shift the intestinal microbiota on rats． Bacteriodetes might have an important role on equol production．

Abstract:Abstract:［Objective］ We characterized eukaryotic community structure and the relationship between the community structure and environmental factors in acidic mine drainage (AMD) lake of a sulfide mine in Anhui Province，China．［Methods］The 18S rRNA gene clone libraries were constructed by using molecular biology techniques to analyze the eukaryotic phylogenetic relationships，and the canonical correspondence analysis (CCA) was used to analyze the relationship between the community structure and environmental factors．［Results］The phylogenetic analysis shows that Ascomycota is widespread in the four samples and dominated in the AMD-1 and AMD-3 clone libraries，whereas Chlorophyta and Basidiomycota are the predominant in AMD-2 and AMD-4 samples，respectively． Moreover，many sequences in libraries were closely related to those of acid-resisting and metal-resisting eukaryotic microbes，such as Sarcinomyces petricola，Penicillium janthinellum，Coniochaeta velutina，Trichoderma viride，Chlorella protothecoides var．acidicola，Ochromonas sp.，and there are high content of known human pathogens，such as Lecythophora hoffmannii，Cryptococcus neoformans． CCA analysis revealed that the critical factors influencing the eukaryotic community structure include TN，SO2－4，Fe2+ and Eh． ［Conclusion］Difference of eukaryotic community structure in time and spare may be related with physico-chemical properties of acidic mine drainage． High content of human pathogens was detected in the acidic ecosystem． The ecological study of eukaryotes under the acidic conditions can help to find efficient methods to process acid mine drainage．

Abstract:Abstract:［Objective］ A protein with insect immunodepressive activity was purified from Xenorhabdus bovienii，a bacterial symbiosis of entomopathogenic nematode． To determine the function of this protein in the pathogenesis of bacterium-nematode symbiosis，we detected the effect of this protein on immune response in Galleria mellonella．［Methods］Proteins from extracellular extact of X． bovienii were purified using ammonium sulfate precipitation，HiTrap Q HP chromatography，HiTrap Butyl FF chromatography，and HiTrap SP HP chromatography． Intra-hemocoel injection assay followed by observation of hemolymph melanization was used for activity determination． Fluorescent microspheres and sepharose beads were used to assess the effect of purified protein on phagocytosis and encapsulation of hemocytes，respectively． The purified protein was identified by 2-D and MS anlysis． The full-length encoding gene was cloned by PCR，and expressed with pET 30a in Escherichia coli． The recombinant protein was purified by Ni-NTA affinity chromatography． ［Results］ An immunodepressive protein was purified and designed as IDP16，which can inhibit the phenoloxidase activity，and weaken the phagocytosis and encapsulation of Galleria mellonella hemocytes． The encoding gene was then cloned and expressed in E． coli． The recombinant protein was also determined to be immunodepressive．［Conclusion］The IDP16 protein from Xenorhabdus bovienii can depress the immune response in insect，which may play an important role in bacteria-host interaction．

Abstract:Abstract:［Objective］To assess the quantification bias associated with incomplete extractions of soil microbial DNA and the feasibility of air-dried soil for microbial ecology study． ［Methods］The flooded rice soil and upland wheat soil were used，and multiple extractions of soil microbial DNA was performed by lysing a single sample for 5 successive times． The copy number of 16S rRNA and amoA genes of Archaea and Bacteria was quantified in each DNA extraction by real-time PCR．［Results］Cumulative DNA yields in 3 successive extractions accounted for more than 76% of microbial DNA in soils，and more than 77. 5% of gene copies could be recovered． Air-drying decreased the abundance of bacterial 16S rRNA gene and archaeal 16S rRNA gene by 90. 3% and 12. 5% ，and the abundance of bacterial and archaeal amoA genes showed a decline by 81. 2% and 84. 3% ，respectively． The decline showed similar trend in two soils，suggesting air-dried soil could be of choice for biogeographic survey of microbial communities． ［Conclusion］Three successive extractions of microbial DNA in soil could be of choice for microbial ecology study in order to reduce quantification bias associated with incomplete DNA recovery． Air-dried soil could be employed under certain circumstances，and further investigation is warranted for the underlying mechanism by which microbial communities manage to survive the desiccation of soil．

Abstract:Abstract:［Objective］To understand the bacterial diversity isolated from the cysts of Heterodera glycines in the soybean field in Heilongjiang Province．［Methods］Bacteria were isolated from cysts on nutrient agar plates using dilution plate method and further identified by phylogenetic analysis based on 16S rDNA gene sequences． ［Results］Totally 90 bacteria strains with different colony morphology were selected on nutrient agar plate and their phylogenetic features were analyzed based on the partial 16S rDNA sequences． In total 7 genera and 22 species were identified，including 46 strains in Gammaproteobacteria (51.1%)，32 in Firmicutes (35.6%)，10 in Betaproteobacteria (11.1%)，and 2 in Alphaproteobacteria (2.2%)． The dominant bacteria species were Pseudomonas and Bacillus．［Conclusion］There was abundant species diversity of bacteria isolated from cysts Heterodera glycines in Heilongjiang，and these bacteria may play a physical and ecological roles in nematodes．

Abstract:Abstract:［Objective］The use of high-temperature Daqu is an important characteristic in Chinese Maotai-flavor liquor making． Thermostable bacteria are predominant in high-temperature Daqu． They play important roles in Maotai-flavor liquor making． Learning the mechanism of thermotolerant performance of bacteria would provide insight into characteristics of this liquor making．［Methods］ We determined the thermotolerance of Bacillus licheniformis CGMCC 3963，and analyzed cell morphology，genomics and transcriptomics．［Results］B． licheniformis CGMCC 3963 could survive at 55℃ and produce many capsules． Genes of class I heat-shock proteins and polyglutamate biosynthesis were both up-regulated．［Conclusion］Much heat-shock proteins and capsule polyglutamate maintained the survival of cell with heat stress．

Abstract:Abstract:［Objective］ Streptomyces sp.X335，harboring a 4.3-kb plasmid pDYM4.3k，was isolated from Tibet sample． Cloning，sequencing，analyzing and functional investigations of pDYM4. 3k．［Methods］Prime walking to obtain plasmid sequence，BLAST to predict gene function，Southern hybridization to determine replication intermediates，and mating to test conjugal function of plasmid．［Results］The complete nucleotide sequence of pDYM4. 3k consisted of 4346 bp，encoding 3 genes of which one resembled Streptomyces major conjugative gene tra and other two were with unknown functions． Experiments demonstrated that a new gene of pDYM4. 3k and its upstream c． 300 bp were required for plasmid replication． Single-stranded pDYM4. 3k as a replication intermediate was detected，indicating it rolling-cirle replication mode． pDYM4. 3k could conjugal transfer among Streptomyces lividans． ［Conclusion］ Streptomyces small plasmid pDYM4. 3k was able to autonomous replication and conjugal transferring．

Abstract:Abstract:［Objective］In order to compare the infection of HIV-1 pseudovirus to suspended and adherent cells，Tzmbl cells containing β-gal ( β-galactosidase ) reporter gene were used here to do the analysis． ［Methods］ HIV-1 pseudoviruses were generated by co-transfection of 293T cells with the plasmid pNL43R-E- and HIV envelope expressing plasmid． Supernatant of co-transfected 293T cells was collected and used to infect Tzmbl cells with or without trypsin treatment． Forty-eight hours after infection，β-gal positive Tzmbl cells and virus infection were determined using X-gal staining and β-glo (β-galactosidase) assay． ［Results］The efficiency of HIV pseudoviruses infection of suspended Tzmbl cell was higher than that of adherent cells and the increase of infection correlated with the pseudoviral subtype．［Conclusion］This study may provide a useful method for HIV biological study and neutralization assays using a singleround replicative pseudovirus in the future．